Cryptococcus neoformans is an environmental fungal pathogen that causes fatal meningoencephalitis and the global burden of the disease is estimated to be annual one million cases with 600,000 death primarily in HIV infected patients. However, the fungus also causes infection in otherwise normal patients at a low frequency. The disease is 100% fatal unless treated and even with the most effective antimycotic agents, the fatality rate is about 25%. C. neoformans is commonly found in the human environment world-wide. In previous years, we discovered that C. neoformans yeast cells invaded the brain by crossing the blood-brain barrier transcellularly. We also, reported that the product of CPS1 gene that plays an important role in association and trversal of C. neoformans yeast cells across the blood-brain barrier (BBB)is hyaluronic acid which is located at the base of the extracellular polysaccharide that surrounds the yeast cells. We also showed, in collaboration with Dr. Jong's group, that protein kinase C-alpha activation is required for the yeast to cross the BBB and CD44 is involved in the association of the yeast cells with the brain microvasular endothelial cells. In order to gain better insight into the invasion process, we have taken a genetic approach during 2008-2009 period. During 2009-2010, in collaboration with Dr. Kim at Johns Hopkins University, we found that the host cell Rac1 GTPase contributes to C. neoformans traversal of the blood-brain barrier and pharmacological inhibition of Rac1 was efficient in inhibiting C. neoformans traversal of the blood-brain barrier, the essential step for the development of C. neoformans meningoencephalitis. This was shown by our demonstrations that (a) Rac1 activation occurs in response to C. neoformans in human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, (b) inhibition of Rac1 was efficient in prevention of HBMEC traversal for both encapsulated and acapsular strains of C. neoformans, and (c) pharmacological inhibition of Rac1 significantly decreased C. neoformans penetration into the brain. These findings demonstrate that targeting host cell signaling molecules essential to C. neoformans traversal of the blood-brain barrier, as shown here with Rac1 GTPase, provides a novel approach to prevention of C. neoformans meningoencephalitis. During 2010-2011,in collaboration with Dr. Jong at the University of Southern California,we found several lines of evidence indicating that C. neoformans invasion is mediated through the endocytic pathway via lipid rafts. Human CD44 molecules from lipid rafts can directly interact with hyaluronic acid, the fungal ligand. Bikunin, which perturbs CD44 function in the lipid raft, can block fungal adhesion and invasion of HBMEC. The lipid raft marker, ganglioside GM1, colocalizes with CD44 on the plasma membrane, and fungal cells can adhere to the host cells where GM1 is enriched. The results suggest the fungal entry is taking place on the lipid rafts. Upon fungal engagement, GM1 was internalized through vesicular structures to the nuclear membrane. This endocytic redistribution process can be perturbed by cytochalasin D, nocodazole, and anti-DYRK3 siRNA. Concomitantly, the knockdown of DYRK3 significantly reduces fungal invasion across the HBMEC monolayer in vitro. Our data demonstrated that the lipid-raft dependent endocytosis process mediates C. neoformans internalization into HBMEC, in which the hosts CD44 protein, cytoskeleton, and intracellular kinase-DYRK3 are involved.During 2011-2012, we investigated the cryptococcal factors that can activate the host signaling pathways that enhance C. neoformans crossing of the blood-brain barrier by using the HBMEC and found that phospholipase secreted by yeast cells of C. neoformans first activates RhoGTPases followed by PKC-alpha, FAK and ezrin. Since cryptococcal urease is reported to be an important factor required for brain invasion and yet the urease activation system has not been studied, we characterized all the factors required for converting apourease into active urease in 2012-2013.Although urea is not required for synthesis of apourease encoded by URE1, the available nitrogen source affected the expression of URE1 as well as the level of the enzyme activity. Activation of the apoenzyme required three accessory proteins,Ure4, Ure6, and Ure7, which are homologs of the bacterial urease accessory proteins UreD, UreF, and UreG, respectively. A yeast two-hybrid assay showed positive interaction of Ure1 with the three accessory proteins encoded by URE4, URE6, and URE7. Metalloproteomic analysis of cryptococcal lysates using inductively coupled plasma mass spectrometry (ICP-MS) and a biochemical assay of urease activity showed that, as in many other organisms, urease is a metallocentric enzyme that requires nickel transported by Nic1 for its catalytic activity. The Ure7 accessory protein (bacterial UreG homolog) binds nickel likely via its conserved histidine-rich domain and appears to be responsible for the incorporation of Ni2 into the apourease. Although the cryptococcal genome lacks the bacterial UreE homolog, Ure7 appeared to combine the functions of bacterial UreE and UreG, thus making this pathogen more similar to that seen with the plant system. Brain invasion by the ure1, ure7, and nic1 mutant strains that lack urease activity was significantly less effective in a mouse model. This indicated that an activated urease and not the Ure1 protein was responsible for enhancement of brain invasion and that the factors required for urease activation in C. neoformans resemble those of plants more than those of bacteria. During the period between 2013 september and 2014 september, we investigated the pattern of urease production in all major four of the major molecular types of C. gattii strains. We found that the URE1 transcriptome as well as the urease levels in C. gattii are less than 50% of those found in C. neoformans strains and the efficiency of HBMEC transcytosis by the C. gattii strains were significantly lower than that of C. neoformans.During late 2014 to early 2015. we have examined the effect of GM-CSF in dissemination of C. neoformans and C. gattii from lung to brain. During the period between 2015 September to 2016, in collaboration with Dr. Jong's group in University of Southern California, we discovered that 14-3-3 gene function of Cryptococcus neoformans is required for growth in the mouse brain and virulence. Starting from January 2017, we have isolated exosomes (microvescicles) from both C. neoformans and C. gattii in order to study their function in brain invasion. Control microvescicles were isolated from non-pathogenic species,Saccharomyces cerevisiae.